Use of erythropoietin-derived peptide through effect on cell damage prevention thereof

ABSTRACT

A peptide is described herein that has: (i) a simple structure compared to existing natural human erythropoietin, thus capable of easily passing through a tissue-blood barrier, (ii) excellent bioactivity with respect to cell-protecting activity, (iii) a low manufacturing cost, thus being economically advantageous, and (iv) no side effects on cell proliferation. Also, a pharmaceutical composition comprising the erythropoietin-derived peptide described herein as an active ingredient is described. The pharmaceutical composition may be used for preventing or treating cell damage-related illnesses, such as stroke, mechanical damage or ischemic damage to the nervous system, myocardial infarction, retinal damage, and diabetes. Also, the described pharmaceutical composition may be used for preventing cell damage.

RELATED APPLICATONS

The present application is a continuation application of InternationalApplication No. PCT/KR2018/002396, filed Feb. 27, 2018, which claimspriority from Korean Patent Application No. 10-2017-0025370, filed onFeb. 27, 2017, the disclosure of which are hereby incorporated byreference herein in their entirety.

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .txt format. The .txtfile contains a sequence listing entitled “16458_7_Seq_Listing_ST25”created on Aug. 23, 2019 and is 8000 bytes in size. The sequence listingcontained in this .txt file is part of the specification and herebyincorporated by reference in its entirety.

BACKGROUND

The present disclosure relates to an erythropoietin (EPO)-derivedpeptide of which a side effect of cell proliferation is eliminated, anda pharmaceutical composition for preventing or treating aneurodegenerative disease, the pharmaceutical composition including theEPO-derived peptide.

During lifetime, the human body is consistently exposed to stimuli,which are harmful to the human body. In response to such exposure, theindividual protects their body. Harmful stimuli include various stimulisuch as hypoxia, infection, mechanical stimulations, etc. Defensemechanisms against such stimuli exist at a cellular level. Variouscytokines that are secreted as a defense mechanism against stimuli playa role in protecting an individual by killing abnormal cells produceddue to exposure to stimuli or by preventing death of normal cells.

Erythropoietin (EPO) is a glycoprotein having a molecular weight ofabout 30,000, and is a hematopoietic cytokine which promotesdifferentiation of red blood cell precursors and increases the number ofred blood cells to exhibit an effect of preventing or improving anemia.This protein initiates its function by binding to a receptor of redblood cell precursors and induces an increase in intracellular calciumions, an increase in DNA biosynthesis, stimulation of hemoglobinproduction, etc. Therefore, EPO may be used as a therapeutic agent foranemia such as anemia in patients with a renal disease, anemia inpremature babies, anemia associated with hypothyroidism, anemiaassociated with malnutrition, anemia associated with chronic renalfailure, postoperative anemia, etc.

Beyond anemia management, EPO has been recently considered as atherapeutic agent for neurological damage. EPO has exhibited tissueprotective ability with respect to nervous system damage, and has alsoexhibited an effect of reducing tissue damage in an animal model ofacute myocardial infarction.

However, in addition to the therapeutic effect on anemia and the abilityto protect nerve cells and nervous tissue, it has been found that anincrease in red blood cells and an increase in platelet activity mayoccur when EPO is injected into the human body. These adverse effectsmay lead to a decrease in the tissue protective ability of EPO.Accordingly, research is being conducted into the development ofmodified EPO or peptides including a partial structure of EPO such asasialo-EPO, carbamylated EPO, EPOtris, EPObis, etc., which are capableof maintaining the tissue protective ability without increasing redblood cells or stimulating platelet activity.

As described above, EPO is known to have a therapeutic effect on anemia,an ability to protect nerve cells or nervous tissue, and an ability toprotect myocardial tissue. EPO is a very active protein, but has a veryhigh production cost. When EPO is injected into peripheral bloodvessels, EPO may not be transported to a target organ due to atissue-blood barrier present in certain target organs, which causesdifficulties in drug delivery. Accordingly, there is a need for aneffective human EPO substitute having low production costs and capableof being easily transported to biological tissues.

Accordingly, the present inventors have prepared a humanerythropoietin-derived peptide having lower production costs thannatural erythropoietin and the ability to easily pass through thetissue-blood barrier in the body while maintaining the cell or tissueprotective abilities of natural human erythropoietin without inducingthe side effect of cell proliferation.

SUMMARY

An aspect provides a peptide, which is described by any one amino acidsequence selected from the group consisting of SEQ ID NOS: 1 to 25.

Another aspect provides a pharmaceutical composition for preventing ortreating a neurodegenerative disease, the pharmaceutical compositionincluding, as an active ingredient, the peptide, one or morepolynucleotides encoding the peptide, a vector including thepolynucleotide, or a host cell including the vector.

Still another aspect provides a method of preventing or treating aneurodegenerative disease, the method including administering thecomposition including, as an active ingredient, the peptide, one or morepolynucleotides encoding the peptide, the vector including thepolynucleotide, or the host cell including the vector.

Still another aspect provides use of the composition including, as anactive ingredient, the peptide, one or more polynucleotides encoding thepeptide, the vector including the polynucleotide, or the host cellincluding the vector in the preparation of a prophylactic or therapeuticagent for a neurodegenerative disease.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D depict graphs showing binding affinities measured by asurface plasmon resonance (SPR) technique to determine whethererythropoietin-derived peptides act on the erythropoietin receptor:

FIG. 1A: determination of binding affinities of ML6-1, ML4-1, ML2-1, andML3-1,

FIG. 1B: determination of binding affinities of ML1-1, ML8-1, ML7-1, andML5-1,

FIG. 1C: determination of binding affinities of ML1, ML1-H1, ML1-H2, andML1-H3, and

FIG. 1D: determination of binding affinities of ML1, ML1-C1, ML1-C2, andML1-C3;

FIGS. 2A-2D depict graphs showing cell protective effects oferythropoietin-derived peptide treatment of cells in which reactiveoxygen species were increased by hydrogen peroxide:

FIG. 2A: effects of treatment with ML1-1, ML4-1, ML6-1, and ML8-1,

FIG. 2B: effects of treatment with ML2-1, ML3-1, ML5-1, and ML7-1,

Control (NGF): cells treated with nerve growth factor (NGF) as a controlgroup,

N.C: cells treated with hydrogen peroxide,

NGF (25 ng/ml): cells treated with NGF after treatment with hydrogenperoxide,

EPO (1 IU): an experimental group treated with 1 IU/ml of naturalerythropoietin after treatment with hydrogen peroxide,

0.25 pM: an experimental group treated with 0.25 pM of the peptide ofthe present disclosure after treatment with hydrogen peroxide,

1 pM: an experimental group treated with 1 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

2 pM: an experimental group treated with 2 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

4 pM: an experimental group treated with 4 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

FIG. 2C: effects of treatment with ML1, ML1-H1, ML1-H2, and ML1-H3,

FIG. 2D: effects of treatment with ML1, ML1-C1, ML1-C2, and ML1-C3,

Control (NGF): cells treated with NGF as a control group,

N.C: cells treated with hydrogen peroxide,

NGF (25 ng/ml): cells treated with NGF after treatment with hydrogenperoxide,

EPO (1 IU): an experimental group treated with 1 IU/ml of naturalerythropoietin after treatment with hydrogen peroxide,

0.25 pM: an experimental group treated with 0.25 pM of the peptide ofthe present disclosure after treatment with hydrogen peroxide,

1 pM: an experimental group treated with 1 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

2 pM: an experimental group treated with 2 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

10 pM: an experimental group treated with 10 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide, and

100 pM: an experimental group treated with 100 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide;

FIGS. 3A and 3B depict graphs showing cell protective effects ofpeptides (ML1-L2, ML1-K2, and ML1-R2) prepared by partially modifyingsequences of an erythropoietin-derived ML1 peptide:

FIG. 3A: cell protective effects in differentiated PC12 cells,

FIG. 3B: cell protective effects in human SH-SY5Y cells,

Control: non-treated cells as a control group,

None: cells treated with hydrogen peroxide,

NGF (25 ng/mL): cells treated with NGF after treatment with hydrogenperoxide, as a positive control group,

EPO (1 IU/mL): an experimental group treated with 1 IU/ml of naturalerythropoietin after treatment with hydrogen peroxide,

Scr: cells treated with 1 pM of scrambled (Scr) peptide after treatmentwith hydrogen peroxide, as a negative control group,

0.1 pM: an experimental group treated with 0.1 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

1 pM: an experimental group treated with 1 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide,

50 pM: an experimental group treated with 50 pM of the peptide of thepresent disclosure after treatment with hydrogen peroxide, and

0.5 nM: an experimental group treated with 0.5 nM of the peptide of thepresent disclosure after treatment with hydrogen peroxide;

FIG. 4 depicts graphs showing cell proliferation rates of peptides(ML1-L2, ML1-K2, and ML1-R2) prepared by partially modifying sequencesof the erythropoietin-derived ML1 peptide:

Control: non-treated cells as a control group,

Scr: cells treated with 1 pM of Scr peptide after treatment withhydrogen peroxide, as a negative control group,

1 pM: an experimental group treated with 1 pM of the peptide of thepresent disclosure,

50 pM: an experimental group treated with 50 pM of the peptide of thepresent disclosure,

0.5 nM: an experimental group treated with 0.5 nM of the peptide of thepresent disclosure,

0.5 IU/mL EPO: an experimental group treated with 0.5 IU/ml of naturalerythropoietin,

1 IU/mL EPO: an experimental group treated with 1 IU/ml of naturalerythropoietin, and

10 IU/mL EPO: an experimental group treated with 10 IU/ml of naturalerythropoietin;

FIG. 5 depicts graphs showing cell proliferative effects of theerythropoietin-derived peptides and peptides prepared by partiallymodifying sequences thereof;

FIG. 6 shows an illustration of a structure of a complex oferythropoietin receptor (EPOR) and erythropoietin (EPO) of oneembodiment and binding target sites; and

FIG. 7 shows an illustration of a substitution process of amino acidsequences of one embodiment.

DESCRIPTION

An aspect provides a peptide, which is described by any one amino acidsequence selected from the group consisting of SEQ ID NOS: 1 to 25.

The peptide may be derived from an erythropoietin protein sequence. Thepeptide may bind to an erythropoietin receptor and may form analpha-helical structure.

The peptide may exhibit cell protective activity and may have no sideeffect of cell proliferation.

The peptide of one embodiment may bind to a target site 1 or a targetsite 2 of an erythropoietin receptor.

The erythropoietin receptor (EPOR) has two target sites, through whichEPOR forms a complex with erythropoietin (EPO). According to previousstudies, of the two binding target sites, the target site 1 forms astrong bond (KD=˜1 nM) and the target site 2 forms a weak bond (KD=˜1μM) (see FIG. 6).

The target site targeted in the present disclosure is a weak bindingsite, and the weak binding of EPOR and the peptide of the presentdisclosure may prevent the side effect (proliferative effect) which isinduced by binding of natural EPO to EPOR thereof. Arg103, Ser104,Leu105, Leu108, and Arg110 are known as crucial amino acid sequences inEOPR, and based on EPO sequences directly binding to these sequences,the target site was determined.

In one embodiment, the present inventors synthesized peptides from apartial target site of natural EPO according to a known solid phasepeptide synthesis technology, and specific characteristics of therespective peptides were identified (see Tables 1 and 2). Further,“LHVDKAVSGLRSLTTL” (SEQ ID NO: 23), which is part of a basic sequence ofML1 was used to prepare peptides having modified amino acids at bothends thereof (see Table 7).

The present inventors confirmed that the peptides of the presentdisclosure may bind to EPOR to exert their actions (see FIG. 1, andTables 8 and 9). The present inventors also confirmed that theerythropoietin-derived peptides prepared in the present disclosure mayform a stable alpha-helix like natural EPO.

The present inventors confirmed that cells under stress environmentsinduced by increased reactive oxygen species due to hydrogen peroxidemay be protected by treatment with the erythropoietin-derived peptideprepared in the present disclosure (see FIGS. 2 and 3). The presentinventors confirmed that inhibition of mitochondrial activity understress environments induced by increased reactive oxygen species due tohydrogen peroxide may be suppressed by treatment with theerythropoietin-derived peptide prepared in the present disclosure (seeFIG. 3). The present inventors confirmed that the peptides (ML1-L2,ML1-K2, and ML1-R2) prepared in the present disclosure have no sideeffect of cell proliferation (see FIG. 4).

Accordingly, the peptides of the present disclosure may bind to EPOR andmay inhibit cell death without the side effect of cell proliferation,thereby being usefully applied as an EPO substitute to the prevention ortreatment with neurodegenerative diseases.

Another aspect provides a pharmaceutical composition for preventing ortreating a neurodegenerative disease, the pharmaceutical compositionincluding, as an active ingredient, the peptide described by any oneamino acid sequence selected from the group consisting of SEQ ID NOS: 1to 25, one or more polynucleotides encoding the peptide, a vectorincluding the polynucleotide, or a host cell including the vector.

The composition may exhibit cell protective activity and may have noside effect of cell proliferation.

The neurodegenerative disease may be selected from the group consistingof stroke, paralysis, myocardial infarction, dementia, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, Huntington's disease,Pick's disease, and Creutzfeldt-Jakob disease.

The host cell may be an HEK-293E cell, a Chinese hamster ovary (CHO)cell, a baby hamster kidney (BHK) cell, an NIH-3T3 cell, an HEK-293Tcell, or a COS-7 cell.

The vector may be selected from the group consisting of a linear DNAvector, a plasmid DNA vector, or a recombinant viral vector. Therecombinant virus may be selected from the group consisting ofretroviruses, adenoviruses, adeno-associated viruses, herpes simplexviruses, and lentiviruses.

A therapeutically effective dose of the composition may vary dependingon various factors, for example, an administration method, a targetarea, a patient's conditions, etc. Thus, when the composition is used inthe human body, the administration dose is required to be suitablydetermined by taking into consideration both safety and efficiency. Itis also possible to estimate the dose for human administration from theeffective dose determined through an animal test. Such considerations tobe taken into the determination of the effective dose are described, forexample, in Hardman and Limbird, eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press;and E. W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

The composition may also include a carrier, a diluent, an excipient, ora combination of two or more thereof, which are commonly used inbiological formulations. The pharmaceutically acceptable carrier is notparticularly limited, as long as it is suitable for in vivo delivery ofthe composition. For example, the compounds described in Merck Index,13th ed., Merck & Co. Inc., physiological saline, sterile water,Ringer's solution, buffered saline, a dextrose solution, a maltodextrinsolution, glycerol, ethanol, and a mixture of one or more of thesecomponents may be used. If necessary, the composition may include othercommon additives such as antioxidants, buffers, bacteriostatic agents,etc.

In addition, the composition may be prepared into injectableformulations, such as aqueous solutions, suspensions, and emulsions,pills, capsules, granules, or tablets, by adding diluents, dispersingagents, surfactants, binders, and lubricants thereto. Furthermore, thecomposition may be appropriately formulated according to each disease orcomponent by a suitable method known in the art or by using a methoddisclosed in Remington's Pharmaceutical Science (Mack PublishingCompany, Easton Pa., 18th, 1990).

The composition may further include one or more active ingredientshaving identical or similar functions. The composition may include theprotein in an amount of 0.0001% by weight to 10% by weight, or 0.001% byweight to 1% by weight, based on the total weight of the composition.

The composition may be administered parenterally (e.g., intravenous,subcutaneous, intraperitoneal, or topical application) or orallyaccording to the intended method. The administration dose may varydepending on a patient's weight, age, sex, health conditions, diet,administration time, administration method, excretion rate, and severityof the disease. A daily dose of the composition may 0.0001 mg/ml to 10mg/ml or 0.0001 mg/ml to 5 mg/ml, and the composition may beadministered once or several times a day.

The vector including the polynucleotide encoding the peptide may beincluded in an amount of 0.05 mg to 500 mg or 0.1 mg to 300 mg, and therecombinant virus including the polynucleotide encoding the peptide ofthe present disclosure may be included in an amount of 10³ IU to 10¹² IU(10 PFU to 10¹⁰ PFU) or 10⁵ IU to 10¹⁰ IU, but is not limited thereto.

Further, the cell including the polynucleotide encoding the peptide maybe included in an amount of 10³ cells to 10⁸ cells, for example, 10⁴cells to 10⁸ cells, 10³ cells to 10⁷ cells, or 10⁴ cells to 10⁷ cells.

With regard to the effective dose of the composition including thevector or cell including the polynucleotide encoding the peptide as anactive ingredient, the vector may be administered in an amount of 0.05mg/kg to 12.5 mg/kg, the recombinant virus may be administered in anamount of 10⁷ viral particles to 10¹¹ viral particles (10⁵ IU to 10⁹IU)/kg, and the cell may be administered in an amount of 10³ cells/kg to10⁶ cells/kg, or the vector may be administered in an amount of 0.1mg/kg to 10 mg/kg, the recombinant virus may be administered in anamount of 10⁸ viral particles to 10¹⁰ viral particles (10⁶ IU to 10⁸IU)/kg, and the cell may be administered in an amount of 10² cells/kg to10⁵ cells/kg twice or three times a day. The composition is notparticularly limited thereto, and may vary depending on a patient'sconditions and development of the neurodegenerative disease.

The composition may further include a carrier, an excipient, and adiluent which are commonly used in the preparation of pharmaceuticalcompositions. The composition may be parenterally administered, and theparenteral administration may be selected from skin externalapplication, intraperitoneal injection, intrarectal injection,subcutaneous injection, intravenous injection, intramuscular injection,and intrathoracic injection, but is not limited thereto.

The composition may be formulated in the form of an externalpreparation, a suppository, and a sterile injectable solution accordingto common methods, respectively. The carrier, the excipient, and thediluent which may be included in the composition may include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Theformulation may be prepared using a commonly used diluent or excipient,such as a filler, an extender, a binder, a wetting agent, adisintegrant, a surfactant, etc. A formulation for parenteraladministration may include a sterilized aqueous solution, a non-aqueoussolvent, a suspension, an emulsion, a lyophilized formulation, and asuppository. The non-aqueous solvent formulation and the suspensionformulation may be propylene glycol, polyethylene glycol, a vegetableoil such as olive oil, or injectable ester such as ethyloleate. A basefor the suppository formulation may be witepsol, macrogol, tween 61,cacao butter, laurin butter, glycerogelatin, etc.

Still another aspect provides a method of preventing or treating aneurodegenerative disease, the method including administering thecomposition including, as an active ingredient, the peptide, one or morepolynucleotides encoding the peptide, the vector including thepolynucleotide, or the host cell including the vector.

An appropriate administration dose of the composition may vary dependingon a patient's conditions and body weight, severity of the disease, atype of a drug, administration route and period, but may beappropriately selected by those skilled in the art. For better effects,the composition may be administered at a dose of 0.0001 mg/kg to 1 g/kgor 0.001 mg/kg to 200 mg/kg per day, but is not limited thereto. Theadministration may be performed once or several times a day. However,the administration dose does not limit the scope of the presentdisclosure in any aspect. Further, the therapeutic agent may beadministered via various routes to mammals such as rats, mice,livestock, humans, etc. All modes of administration are contemplated,for example, administration may be made orally, rectally, or byintravenous, intramuscular, subcutaneous, intradural, orintracerebroventricular injection.

Still another aspect provides use of the composition including, as anactive ingredient, the peptide, one or more polynucleotides encoding thepeptide, the vector including the polynucleotide, or the host cellincluding the vector in the preparation of a prophylactic or therapeuticagent for a neurodegenerative disease.

The peptide of the present disclosure may bind to EPOR and may inhibitcell death without the side effect of cell proliferation, thereby beingusefully applied as an EPO substitute to the prevention or treatment ofa neurodegenerative disease.

Advantageous Effects of Disclosure

A peptide according to an aspect has a simple structure, as comparedwith natural human erythropoietin, and thus the peptide easily passesthrough a tissue-blood barrier, exhibits excellent physiologicalactivity due to cell protective effects, and is economicallyadvantageous due to its low production cost. Further, the peptide has noside effects of cell proliferation, and thus a pharmaceuticalcomposition including the described erythropoietin-derived peptide of anaspect as an active ingredient may be usefully applied to the preventionor treatment of cell damage-related illnesses, such as stroke,mechanical damage or ischemic injury to the nervous system, myocardialinfarction, retinal damage, diabetes, etc., and the prevention of celldamages.

EXAMPLES

Hereinafter, exemplary embodiments will be provided for betterunderstanding of the present disclosure. However, the followingexemplary embodiments are provided only for understanding the presentdisclosure more easily, but the content of the present disclosure is notlimited thereby.

Example 1. Synthesis of Erythropoietin-Derived Peptides

Erythropoietin-derived peptides of the present disclosure weresynthesized as monomers according to a known solid phase peptidesynthesis technology (Peptron, Daejeon, Korea).

In detail, erythropoietin-derived peptides, which are able to bind tocrucial amino acid sequences (Arg103, Ser104, Leu105, Leu108 and Arg110)in a sequence of a target site (site 2) of the natural erythropoietinreceptor were synthesized, and specific characteristics of the peptideswere examined, respectively. To determine concentrations of thesynthesized peptides, liquid chromatography/mass-selective detector (HP1100 series) was used. Purity was measured by high performance liquidchromatography (SHIMADZU prominence HPLC) (>95% purity). Theerythropoietin-derived peptides are shown in Table 1 below.

TABLE 1 Pep- SEQ Pep- SEQ tide ID tide ID name Sequence NO: nameSequence NO: ML1 LQLHVDKAVSGLRSLTTL 1 ML1-1 LQLHVLKRVSGLLS  9 LRALGHTMLLKALG ML2 LHVDKAVSGLRSLTTLLR 2 ML2-1 RHVQKAESGLRSLT 10 AL KLLREL ML3TKVNFYAWKR 3 ML3-1 TRVNYQAWKR 11 ML4 DKAVSGLRSLTTLLRALG 4 ML4-1KKAVSGLKTLTHIL 12 AQKEAI RALGAQKEAI ML5 SGLRSLTTLLRALG 5 ML5-1AGLRSRAHLRRALA 13 ML6 SGLRSLTTLLRALGAQKE 6 ML6-1 KGLRSLISLLRALG 14 AIAQKEAI ML7 WEPLQLHVDKAVSGLRSL 7 ML7-1 DEALDLEVDKAATG 15 TTLLRALLRTLTTLIRAL ML8 DKAVSGLRSLTTLLRAL 8 ML8-1 NKAVAGLRSLTVN 16

Hydrophobicity, charge, and isoelectric point (pi) of theerythropoietin-derived peptides, ML1-1, ML2-1, ML3-1, ML4-1, ML5-1,ML6-1, ML7-1, and ML8-1 were calculated and shown in Table 2 below.

TABLE 2 Peptide name Hydrophobicity Charge(pH 7) pl Target site ML1-18.25 3.4 11.2 2 ML2-1 −4.45 3.2 10.94 2 ML3-1 −10.07 2.9 10.94 1 ML4-1 56.1 11.41 2 ML5-1 −4.15 4.1 12.48 2 ML6-1 8.85 2.9 10.94 2 ML7-1 2.05−2.1 4.59 2 ML8-1 5.7 1.9 11.12 2

Example 2. Synthesis of Erythropoietin-Derived Peptides Using PartialSequence (1)

For sequence modification experiments, a binding model of erythropoietinand its receptor was based on a previously known binding structure(Protein Data Bank ID: 1 EER). Based on known characteristics of aminoacids, amino acids of the erythropoietin-derived peptides weresubstituted. Amino acids are classified into 4 types ({circle around(1)} non-polar or hydrophobic, {circle around (2)} neutral, {circlearound (3)} negatively charged, {circle around (4)} positively charged)according to polarity of their side chains. Based on information ofnon-polar (hydrophobic), neutral, negatively charged, or positivelycharged amino acids, the existing amino acid sequences were substitutedto induce modification in respective characteristics.

Peptides prepared by partially modifying sequences of ML1 peptide andtheir characteristics are shown in Tables 3 and 4.

TABLE 3 Peptide  SEO ID name Sequence NO: ML1 LQLHVDKAVSGLRSLTTLLRALG  1ML1-H1 LQLHVLKAVSGLLTHTTLLKALG 17 ML1-H2 LQLHVLKAVSGLLTLTMIRRALG 18ML1-H3 LQLHVLKAVAGLRTLAMIRRALA 19

TABLE 4 Num- ber Iso- Net Peptide of Molecular Absorption electriccharge Predicted name residue weight coefficient point (pH 7) solubilityML1 23 2461.9 0 M⁻¹cm⁻¹ pH 11.23 2.1 Low g/mol solubility in waterML1-H1 23 2426.94 0 M⁻¹cm⁻¹ pH 10.73 2.2 Low g/mol solubility in waterML1-H2 23 2504.09 0 M⁻¹cm⁻¹ pH 12.13 3.1 Low g/mol solubility in waterML1-H3 23 2515.12 0 M⁻¹cm⁻¹ pH 12.41 4.1 Low g/mol solubility in water

Example 3. Synthesis of Erythropoietin-Derived Peptides Using PartialSequence (2)

Partial sequences of the peptides were substituted using the basicsequence of ML1 as in Example 2. In this regard, amino acids weresubstituted based on the existing binding model of erythropoietin andits receptor without hindering the existing binding structure (adistance between proteins or a protein structure). FIG. 7 illustratesexemplary substitution of amino acid sequences. Since substitution ofalanine (Ala) with arginine (Arg) hinders the existing binding,substitution with serine (Ser) may be performed to prevent hindrance ofthe binding.

Peptides prepared by modifying the charge of the ML1 peptide andcharacteristics thereof are shown in Tables 5 and 6 below.

TABLE 5 Peptide  SEQ ID  name Sequence NO: ML1-C1LDLEVDKAVSGLRSLTTLLRALG 20 ML1 LQLHVDKAVSGLRSLTTLLRALG  1 ML1-C2LQRHVDKRVSGLRSLTTLLRALG 21 ML1-C3 LQRHVKKRVKGLKSLTTLLRALG 22

TABLE 6 Num- ber Iso- Net Peptide of Molecular Absorption electriccharge Predicted name residue weight coefficient point (pH 7) solubilityML1-C1 23 2440.83 0 M⁻¹cm⁻¹ pH 6.96 0 High g/mol solubility in water ML123 2461.9 0 M⁻¹cm⁻¹ pH 11.23 2.1 Low g/mol solubility in water ML1-C2 232590.04 0 M⁻¹cm⁻¹ pH 12.12 4.1 High g/mol solubility in water ML1-03 232616.21 0 M⁻¹cm⁻¹ pH 12.45 7.1 High g/mol solubility in water

Example 4. Synthesis of Erythropoietin-Derived Peptides Using PartialSequence (3)

A partial sequence “LHVDKAVSGLRSLTTL” (SEQ ID NO: 23) of the ML1 basicsequence was used to prepare peptides having modified amino acids atboth ends thereof, as in Table 7 below.

TABLE 7 Peptide SEO ID  name Sequence NO: ML1-L2 L HVDKAVSGLRSLTT L 23ML1-K2 K HVDKAVSGLRSLTT K 24 ML1-R2 R HVDKAVSGLRSLTT R 25

Experimental Example

I. Determination of Binding Affinity of Erythropoietin-Derived Peptideto Erythropoietin Receptor (EPOR)

To determine whether the erythropoietin-derived peptides prepared inExamples 1 to 3 are able to bind to the erythropoietin receptor havingthe target site to exert their actions, a surface plasmon resonance(SPR) technique was used to determine binding affinity. The SPRtechnique is to measure interactions between biomolecules in real-timeby using an optical principle without specific labeling, and is a systemanalyzing affinity between two molecules and kinetics, i.e., anassociation rate (Ka) and a dissociation rate (Kd).

In detail, real-time SPR analysis may be performed using Reichert SPRBiosensor SR 7500C instrument (Reichert Inc., NY, USA). Soluble mouseEPOR chimera proteins (R&D Systems, Minneapolis, Minn., USA) werecovalently linked to a carboxymethylated dextran matrix-coated chip(BR-1005-39, Pharmacia Biosensor AB) by an amine coupling procedureusing an amine coupling kit (BR-1000-50, GE Healthcare, USA) inaccordance with manufacturer's instructions. Each 5 μM, 2.5 μM, and 1.25μM of the peptide samples of the present disclosure and scrambledpeptides were applied at a flow rate of 5 μl/minute, and the experimentswere independently performed in duplicate. For signal normalization,DMSO was applied at a flow rate of 5 μl/minute. After each bindingcycle, the sensor chip was regenerated by injecting 25 mM of acetic acidat a flow rate of 20 μl/minute.

As a result, as shown in FIG. 1, the result values were increasedaccording to the concentrations of the erythropoietin-derived peptidesof one embodiment, and thus it was confirmed that theerythropoietin-derived peptides bind to the erythropoietin receptorhaving the target site to exert their actions (FIG. 1). Further, asshown in Tables 8 and 9, it was also confirmed that theerythropoietin-derived peptides of one embodiment exhibited bindingaffinities similar to the known binding affinity (˜1 uM).

TABLE 8 Ka Kd KD ML1 1.311 × 10³ 8.5 × 10⁻³ 6.46077 μM ML2  1.6 × 10 4.4 × 10⁻³    273 μM ML3 2.05 × 10²   3 × 10⁻³ 14.6341 μM ML4  2.2 × 10²2.2 × 10⁻³    10 μM ML5 3.04 × 10² 0.1 × 10⁻³ 0.32894 μM ML6  5.0 × 10 4.5 × 10⁻²    900 μM ML7 3.00 × 10² 0.2 × 10⁻²  6.666 μM ML8  1.8 × 10²0.8 × 10⁻¹  444.44 μM ML1-1 5.55 × 10³ 5.9 × 10⁻³   1.06 μM ML2-1  3.1 ×10² 4.1 × 10⁻³   14.3 μM ML3-1 3.08 × 10³ 1.3 × 10⁻²   4.31 μM ML4-14.10 × 10² 1.20 × 10²    39.34 mM ML5-1 4.42 × 10² 3.46 × 10⁻²   78.28μM ML6-1  1.9 × 10²   3 × 10⁻²  157.8 μM ML7-1 2.26 × 10² 1.44 × 10⁻²  63.70 μM ML8-1  6.4 × 10  1.5 × 10⁻¹  2.37 mM

TABLE 9 Km Ka Kd KD ML1 1.26E+05 1310.8 8.47E−03 6.46077 μM ML1-H14.79E+05 1.01E+03 7.94E−03 7.84542 μM ML1-H2 1.00E+10 3434.3 1.05E−03306.977 nM ML1-H3 1.00E+10 4157.6 4.77E−03 1.14651 μM ML1-C1 9.23E+051745.7 0.2617 149.921 μM ML1-C2 4.58E+05 1.59E+03 0.01876 11.7609 μMML1-C3 1.46E+05 1104.9 0.01086 9.82836 μM

In other words, it was confirmed that the peptides according to specificembodiments are those derived from the erythropoietin binding site, andthus they have binding affinity to the erythropoietin receptor.

Determination of secondary alpha-helix formation oferythropoietin-derived peptide

The present inventors determined whether the erythropoietin-derivedpeptides synthesized in Example 1 are able to form a stable alpha-helix,like natural erythropoietin.

As a result, it was confirmed that the erythropoietin-derived peptidessynthesized in Example 1 formed a stable secondary alpha-helix, likenatural erythropoietin.

II. Determination of Cell Protective Effect of Erythropoietin-DerivedPeptide (1)

To determine whether the erythropoietin-derived peptides prepared inExamples 1 to 3 exhibit cell protective effects, cell viability wasdetermined under stress conditions where an increase in reactive oxygenspecies was induced by hydrogen peroxide (H₂O₂).

In detail, to evaluate cell viability, an MTS assay (CellTiter 96Aqueous One Solution Cell Proliferation Assay, Promega, Madison, Wis.,USA) was performed. PC12 cells were seeded in a 96-well plate (5×10⁴cells per well), and an increase in reactive oxygen species was inducedusing 150 μM of hydrogen peroxide (H₂O₂). Thereafter, 25 ng/ml of nervegrowth factor (NGF) was added as a positive control group, and 1 IU/mlof the erythropoietin compound, 0.25 pM, 1 pM, 2 pM, or 4 pM of thepeptide of Example 1, each 0.25 pM, 1 pM, 2 pM, 10 pM, or 100 pM of thepeptides of Example 2 and 3, or 0.1 pM, 1 pM, 50 pM, or 0.5 nM of thepeptide of Example 4 was added, and 20 μl of an MTS solution was addedto each well, and left for 3 hours. The initial number of cells (0 hour)and the number of cells after 48 hours were counted. Intracellularsoluble formazan produced by cell reduction was determined by recordingabsorbance of each 96-well plate at a wavelength of 490 nm using a VERSAMAX.

As a result, as shown in FIG. 2, it was confirmed that theerythropoietin-derived peptides protected cells from cell death causedby the increase in reactive oxygen species (FIG. 2). This result wassimilar to the cell protective effect by treatment with the naturalerythropoietin compound.

III. Determination of Cell Protective Effect of Erythropoietin-DerivedPeptide (2)

To determine whether the erythropoietin-derived peptide prepared inExample 4 exhibits the cell protective effect, mitochondrial activitywas determined under stress conditions where an increase in reactiveoxygen species was induced by hydrogen peroxide (H₂O₂).

In detail, PC12 cells or human SH-SY5Y cells were seeded in a 96-wellplate (5×10⁴ cells per well), and an increase in reactive oxygen specieswas induced using 150 μM of hydrogen peroxide (H₂O₂). Thereafter, 25ng/ml of NGF was added as a positive control group, 1 IU/ml of theerythropoietin compound, or 0.1 pM, 1 pM, 50 pM, or 0.5 nM of thepeptide of Example 4 were added.

When mitochondrial activity is suppressed, mitochondrial swelling due toabnormalities of the mitochondrial membrane potential, dysfunction dueto oxidative stress such as reactive oxygen species or free radicals,dysfunction due to genetic factors, and dysfunction due to defects inoxidative phosphorylation for mitochondrial energy production occur.Thus, mitochondrial activity may be determined by measuring themitochondrial membrane potential. Tetramethylrhodamine methyl ester(TMRM) staining of mitochondria was performed. Since TMRM stainingintensity is increased in proportion to the mitochondrial membranepotential, the intracellular mitochondrial membrane potential wasdetermined by measuring the TMRM staining intensity using a microplatereader (excitation, 485 nm; emission, 535 nm).

As a result, as shown in FIG. 3, it was confirmed that theerythropoietin-derived peptides suppressed inhibition of mitochondrialactivity caused by increased reactive oxygen species (FIG. 3). Thisresult was similar to the effect by treatment with the naturalerythropoietin compound.

IV. Determination of Cell Proliferation-Inhibitory Effect ofErythropoietin-Derived Peptide (1)

Side effects such as cell proliferation were determined for the threepeptides (ML1-L2, ML1-K2, and ML1-R2) prepared in Example 4.

In detail, to determine cell proliferation degree, an MTS assay(CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega,Madison, Wis., USA) was performed. PC12 cells were seeded in a 96-wellplate (5×10⁴ cells per well), and 1 pM of scrambled peptide (Scr) as anegative control group, 0.5 IU/ml, 1 IU/ml, or 10 IU/ml of theerythropoietin compound, or 1 pM, 10 pM, or 0.5 nM of the peptide ofExample 4 was added, and 20 μl of an MTS solution was added to eachwell, and left for 3 hours. The initial number of cells (0 hour) and thenumber of cells after 48 hours were counted. Intracellular solubleformazan produced by cell reduction was determined by recordingabsorbance of each 96-well plate at a wavelength of 490 nm using a VERSAMAX.

As a result, as shown in FIG. 4, it was confirmed that all the peptidesshowed cell proliferation rates similar to that of the control group,and they showed no side effect of cell proliferation.

V. Determination of Cell Proliferation-Inhibitory Effect ofErythropoietin-Derived Peptide (2)

To determine the side effect of cell proliferation with respect to thepeptides prepared in Examples 1 to 3, cell viability was evaluated by anMTT assay.

In detail, PC12 cells were cultured in a DMEM (Dulbecco's ModifiedEagle's Medium) medium (Hyclone, USA) and an RPMI1640 medium (Hyclone,Utah, USA) each supplemented with 10% fetal bovine serum (FBS, Hyclone,Utah, USA), 100 unit/ml penicillin, and 100 μg/ml streptomycin (Hyclone,Utah, USA) in an incubator under conditions of 5% CO₂ and 37° C. PC12cells were seeded in a 96-well culture plate at a density of 5×10⁴cells/ml, and cultured under conditions of 37° C. and 5% CO₂ for 24hours. Thereafter, the cells were treated with each of the peptides ofExamples 1 to 3, which were prepared at a concentration of 10 ng/ml,followed by incubation for 24 hours. Thereafter, 20 μl of 5 mg/ml3-[4,5-dimethyl-thiazol]-2,5-diphenyl-tetrazolium bromide (MTT) reagentwas added thereto, and allowed to react for 2 hours. After reaction, 200μl of dimethyl sulfoxide (DMSO, Duksan, Gyeonggi-do, Korea) was addedthereto to completely dissolve formed formazan, and absorbance at 570 nmwas measured using a microplate reader (Molecular Devices, CA, USA).

As a result, as shown in FIG. 5, it was confirmed that all the peptidesshowed cell proliferation rates similar to that of the control group,and they showed no side effect of cell proliferation.

The foregoing description of the present disclosure is for illustrativepurposes only, and those of ordinary skill in the art readily understandthat the present disclosure may be implemented in a different specificform without changing the technical spirit or essential characteristicsthereof. It is therefore to be understood that the above-describedembodiments are not limitative, but illustrative in all aspects.

1. A peptide described by any one amino acid sequence selected from thegroup consisting of SEQ ID NOS: 1 or
 9. 2. The peptide of claim 1,wherein the peptide is derived from an erythropoietin protein sequence.3. The peptide of claim 1, wherein the peptide binds to anerythropoietin receptor.
 4. The peptide of claim 1, wherein the peptideforms an alpha-helical structure.
 5. The peptide of claim 1, wherein thepeptide exhibits cell protective activity.
 6. The peptide of claim 1,wherein the peptide has no side effect of cell proliferation.
 7. Apharmaceutical composition for preventing or treating aneurodegenerative disease, the pharmaceutical composition comprising, asan active ingredient, the peptide of claim 1, one or morepolynucleotides encoding the peptide, a vector comprising thepolynucleotide, or a host cell comprising the vector.
 8. Thepharmaceutical composition for preventing or treating aneurodegenerative disease of claim 7, wherein the neurodegenerativedisease is any one selected from the group consisting of stroke,paralysis, myocardial infarction, dementia, Alzheimer's disease,Parkinson's disease, multiple sclerosis, Huntington's disease, Pick'sdisease, and Creutzfeldt-Jakob disease.
 9. The pharmaceuticalcomposition for preventing or treating a neurodegenerative disease ofclaim 7, wherein the host cell is an HEK-293E cell, a Chinese hamsterovary (CHO) cell, a baby hamster kidney (BHK) cell, an NIH-3T3 cell, anHEK-293T cell, or a COS-7 cell.
 10. The pharmaceutical composition forpreventing or treating a neurodegenerative disease of claim 7, whereinthe vector is a linear DNA vector, a plasmid DNA vector, or arecombinant viral vector.
 11. The pharmaceutical composition forpreventing or treating a neurodegenerative disease of claim 10, whereinthe recombinant virus is any one selected from the group consisting ofretroviruses, adenoviruses, adeno-associated viruses, herpes simplexviruses, and lentiviruses.
 12. A method of preventing or treating aneurodegenerative disease, the method comprising: administering acomposition comprising, as an active ingredient, the peptide of claim 1,one or more polynucleotides encoding the peptide, a vector comprisingthe polynucleotide, or a host cell comprising the vector.
 13. Use of acomposition comprising, as an active ingredient, the peptide of claim 1,one or more polynucleotides encoding the peptide, a vector comprisingthe polynucleotide, or a host cell comprising the vector in thepreparation of a prophylactic or therapeutic agent for aneurodegenerative disease.